Methotrexate is of proven utility in the therapy of human neoplasms in low dose regimens and in high dose regimens combined with leucovorin (CF) rescue; a new treatment modality in which high dose methotrexate is combined with thymidine rescue is currently in Phase I clinical trial. This investigation will study (1) the folate biochemistry underlying the cytotoxicity and selectivity of low dose methotrexate, (2) the importance of the metabolism of CF to high dose methotrexate/CF rescue, (3) the mechanism of thymidine reversal of MTX in vitro and in vivo, and (4) the folate and nucleotide metabolism concerned with the antimetabolite selectivity of these treatment modalities. Chromatographic analysis of the distribution of intracellular folates among the polyglutamyl tetrahydrofolate cofactor forms following exposure to MTX and during rescue will allow the biochemical basis of such antifolate modalities to be directly examined. Use of new analytical methods which are proposed herein for the measurement of tetrahydrofolate and dihydrofolate and our recently developed techniques for assay of tumor levels of deoxyuridylate and thymidylate synthetase offer a unique approach to these questions. This study also seeks to identify key biochemical predictors of tumor sensitivity in rodent tumors and human tumors growing in nude mice and to evaluate these parameters in pediatric neoplasms as diagnostic aids for the rational pre-selection of sensitive tumors.